ANS 3319C Reproductive Physiology and Endocrinology
Techniques for In-Vitro Embryo Production
Objectives
1) To gain an understanding of the process of in-vitro embryo production in cattle
and other domestic species.
2) To provide hands-on experience in the techniques involved in in-vitro embryo
production including oocyte aspiration and collection, maturation, fertilization, and
embryo culture.
Application of In-Vitro Embryo Technologies to Domestic Animal Species
1) Production of embryos from genetically valuable animals that are either infertile
or that are deceased.
2) An alternative means to produce embryos from valuable animals rather than
using superovulation
3) An inexpensive method for producing embryos using abattoir-derived ovaries
Overview
Although the production of domestic animal embryos in vitro is still a relatively new
technique in commercial and clinical settings, work on in vitro fertilization began as early
as the 1930’s with rabbit oocytes. While these first attempts at in vitro fertilization were
not successful, subsequent research in the late 1950’s led to the birth of rabbit pups
produced using oocytes fertilized in vitro. In the late 1960’s, work with human oocytes
led to the birth of the first baby, Louise Brown (Figure 1), following in vitro fertilization in
the United Kingdom in 1978. Since this time the production of human preimplantation
embryos in vitro has become a common treatment for infertility. It is estimated that over
100,000 babies have been born using this technique in the United States alone.
In terms of in vitro embryo techniques in domestic animals, the first calf (Figure 1),
lambs and pigs produced following in vitro fertilization were born in the early 1980’s and
the live birth of a foal following in vitro fertilization was reported in the early 1990’s.
The production of bovine embryos in vitro has been the most successful of all the
domestic animal species. Research on this topic in the 1980’s led to improved in vitro
maturation medium and also techniques for capacitation of sperm in vitro. In the 1990’s
new developments led to improved embryo culture conditions such that perimplantation
bovine embryos could be cultured to the blastocyst stage in vitro. Over the last 10-15
years, the production of bovine embryos in vitro for commercial use has increased
significantly and now there are several hundred thousand bovine in vitro produced
embryos transferred worldwide each year. The production of embryos in vitro of other
domestic species, especially the pig and the horse, have been less successful and
further research is necessary before these techniques can be applied efficiently in
commercial settings.
This handout was developed by former Animal Sciences graduate students Jeremy Block and Luiz Augusto (Guto) de
Castro e Paula. Jeremy and Guto received their PhD in reproductive physiology in the Department of Animal Science
at the University of Florida. This handout was prepared using material from Dr. Pete Hansen’s web page on
production of bovine embryos http://www.animal.ufl.edu/hansen/IVF/default.htm.
ANS 3319C Reproductive Physiology & Endocrinology – Techniques for In vitro Embryo Production
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Although there is variation among different species in the exact procedures for in-vitro
embryo production, in general the procedure involves 4 major steps: 1) the collection of
immature oocytes, 2) the maturation of immature oocytes, 3) the fertilization of mature
oocytes and 4) the culture of embryos. Each of these steps will be discussed in more
detail in the subsequent sections.
Figure 1. Shown is the first baby born following in vitro fertilization, Louise Brown, in 1978 (left panel) and
first calf born following in vitro fertilization, named Virgil, in 1981 (right panel).
Oocyte Collection
Immature oocytes in the form of cumulus-oocyte complexes (COC; figure 3) can be
collected from live donor animals or directly from ovaries obtained from a abattoir.
There are 2 main ways in which oocytes can be collected from live donors, 1) surgically
by laparotomy and aspiration using a syringe and needle (Figure 2) or 2) using
transvaginal ovum-pick-up techniques as shown in Figure 2.
In the case of ovaries obtained from a abattoir or a deceased donor animal, the ovaries
are generally transported to the laboratory in physiological saline that contains some
form of antibiotic to help prevent bacterial contamination at approximately 22-24 ºC.
The best results are obtained when oocytes are collected within 4-6 hrs after slaughter.
Oocytes can be collected from abattoir derived ovaries either by aspiration using a
syringe and needle or by “slashing” the surface of the ovary with a scalpel blade and
collection of oocytes into a beaker as shown in figure 3.
Oocyte Maturation
Following collection, cumulus-oocyte complexes are washed several times and then
placed into maturation medium for a specified amount of time depending on the species
(Table 1). This process is meant to mimic what occurs in-vivo following the LH surge.
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Thus during this culture period, the oocyte will resume meiosis and arrest at metaphase
II so that it is ready for fertilization. The COC also undergoes other morphological
changes during maturation, including the expansion of the cumulus cells (Figure 3). In
some cases, such as in humans and horses, it is more common to allow oocytes to
mature in vivo and then collect them for in vitro fertilization and embryo culture.
Figure 2. Oocyte collection using ultrasound-guided transvaginal aspiration (left panel) and using a
syringe and needle (right panel).
Figure 3. Collection of oocytes by “slashing” the ovarian surface (left panel) and several immature bovine
cumulus-oocyte complexes following collection (right panel).
Although the mammalian oocyte resumes meiosis immediately after removal from the
follicular environment it is important to place the COC into maturation medium as soon
as possible following collection to provide an optimal microenvironment for oocyte
maturation. The typical maturation medium will include several components including
nutrients (pyruvate, glucose, glutamine, serum), hormones and gonadotrophins
(estrogen, LH, FSH), and antibiotics (penicillin/streptomycin or gentamicin).
ANS 3319C Reproductive Physiology & Endocrinology – Techniques for In vitro Embryo Production
Species
Duration of oocyte maturation
Cow
Pig
Horse
Human
Mouse
21-24 hrs
40-44 hrs
24-48 hrs
28-36 hrs
16-17 hrs
4
Table 1. Duration of in vitro oocyte maturation in various species.
Collected COC are generally matured in 50 μL microdrops (10 COC/drop) or in wells of
a 4-well plate (40-50 COC/well; Figure 4) overlaid with mineral oil to help prevent
evaporation of the maturation medium. Once placed into maturation drops, COC are
placed in an incubator set at 37-39 ºC for the desired amount of time depending on the
species (Table 1).
Figure 4. Petri dishes typically used for in vitro maturation (left panel) and mature bovine cumulus-oocyte
complexes following 22-24 hrs of in vitro maturation (right panel). Note the expansion of the cumulus
cells.
In vitro fertilization
For in vitro fertilization to occur, the media used must be capable of supplying the sperm
cells with nutrients and chemical signals to enhance sperm motility and induction of
capacitation, to facilitate the fusion of the gametes and the beginning of embryonic
development. The in vitro fertilization process can be divided in three main steps:
a. COC washing
Necessary so that hormones, nutrients and metabolites present in the maturation
microdrop are not carried over to the fertilization drop;
ANS 3319C Reproductive Physiology & Endocrinology – Techniques for In vitro Embryo Production
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Procedure
1. Transfer COCs from each maturation microdrop to the X-plate containing the
buffer HEPES-TALP;
2. Transfer 10 COCs from the X-plate to each well of the 4-well fertilization plate;
b. Sperm purification
Necessary so that sperm cells can be washed from the extender + criopreserver (if
frozen is used)/seminal plasma (if fresh semen is used), selected for alive motile
sperm cells and provided with nutrients, buffers and chemical signals to induce
capacitation and hyperactivation.
Procedure
1.
2.
3.
4.
Place 1.5 ml of 90% Percoll and 1.5 ml of HEPES-TL to one 15 ml conical tube;
Mix to make a solution of 45% Percoll;
In another 15 ml conical tube, add 3 ml of 90% Percoll;
Make a Percoll gradient (45% over 90%) by slowly layering the 90 % Percoll on
the bottom of the tube containing the 45% Percoll using a plastic Pasteur pipet;
5. If using frozen semen (which is usually the case in IVF of domestic animal
species), thaw enough straws of semen in a citothaw for 45-60 seconds or in
another thermo with water pre-warmed to 37oC;
6. Wipe the semen straw dry with a kimwipe, cut the tip of the straw with a scissors
and expel contents of the straw onto the top of the Percoll gradient (Figure 5);
Care must be taken so that the gradient is not disturbed and the semen lie on top
of the 45% layer;
Figure 5.. Layering of sperm onto Percoll. After cutting the tip of the straw (Left panel), the
contents of the straw are expelled onto the top of the Percoll gradient (right panel). Here,
removal of the semen is facilitated by using a homemade plunger.
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7. Place the conical tube containing the semen and Percoll gradient into a
centrifuge carrier that has been pre-warmed to 38.5°C, and centrifuge at 1000 x
g for 10 min;
8. After centrifugation, collect sperm pellet from the bottom of the conical tube
(Figure 6);
Figure 6. Removal of sperm from the bottom of the Percoll gradient.
9. Place the sperm pellet into a 15 ml conical tube containing 10 ml Sp-TALP and
place in a warm centrifuge carrier before centrifuging for 5 min at 200 x g;
10. Remove the supernatant with a Pasteur pipet while being careful not to disturb
the pellet (Figure 7);
Figure 7. Washing sperm in Sp-TALP. The left panel shows the washed and centrifuged sperm.
The right panel shows the pellet of sperm remaining in the tube after aspiration of the
supernatant.
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11. Determine dilution required to bring sperm to a concentration of 26 x 106/ml (this
will produce a final concentration of sperm in the fertilization drop of 1 x 106/ml)
using a hemocytometer. Use IVF-TALP media to dilute the sperm solution
(Usually 2 ml of IVF-TALP is enough to bring the content of 3 semen straws to
the desired 26 x 106 sperm cells/ml);
c. Fertilization
At this point, sperm cells can be added to the wells containing the COCs so that
fertilization can take place.
Procedure
1.
Add 25 µl sperm preparation (the IVF-TALP contains heparin which will
help in capacitation of the sperm cells);
2.
25 µl PHE mix into each well (PHE is the acronym for penicillamine,
hypotaurine and epinephrine which are molecules know to induce sperm
hyperactivation);
3.
Place the 4-well fertilization plates in a incubator (5% CO2 in air) at 38.5oC
for 8-20 h;
Alternative in vitro fertilization technique: Intracytoplasmatic sperm injection (ICSI)
In certain circumstances, fertilization can be accomplished using ICSI. ICSI is widely
used in humans when the male has poor sperm quality (low concentration, motility etc.),
although, in some clinics, this is a standard procedure regardless of sperm quality. In
this techinique, fertilization is assisted by injecting one selected sperm cell into one
mature oocyte. ICSI is also generally used as the in vitro fertilization procedure in
horses (See Figure 8).
Figure 8 . Immobilizing the sperm's tail before picking it up (left), injection of sperm into the egg (middle)
and fertilized egg demonstrating the two nuclei--one from the father, one from the mother (right). Picutres
from http://www.infertile.com/treatmnt/treats/icsi.htm.
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Embryo culture
Embryo culture is the step that follows fertilization. During this stage, the newly formed
zygote will need to be provided with the conditions necessary to start dividing and grow
in the most similar way to what would be expected to be happening in the first few days
of pregnancy. There are several types of embryo culture:
a. In vivo – fertilized oocytes are placed in the oviduct of a synchronized female
and grown for 6-8 days. The embryos are then retrieved and transferred to its
definitive mother. In some cases, this type of culture can be performed across
species, e.g., bovine or equine embryos can be successfully cultured in the
sheep oviduct;
b. Co-culture – fertilized oocytes are placed in culture drops containing cells from
the oviduct to try to mimic the maternal environment;
c. Semi-defined medim – fertilized oocytes are placed in culture drops containing
serum. It is called semi-defined medium because the composition of the serum is
variable and usually unknown. This is the most common method of embryo
culture in domestic species;
d. Defined medium – fertilized oocytes are placed in culture drops where
concentrations of all the components are known, including growth factors;
e. Sequential culture – the embryonic needs change as it grows. The idea of
sequential culture is to place embryos in culture medium containing the nutrients
necessary for one specific stage of development, mimicking the maternal
environment;
Two of the most common culture media used in embryo culture are SOF (Synthetic
Oviduct Fluid) and KSOM Potassium Simplex Optimized Medium). Theses medias
contain such items as nutrients (carbohydrates, amino acids) buffers, and antibiotics.
Other factors affecting the embryonic development in vitro:
a. Gas phase – embryo culture can be done using determined concentration of
gases. The two most commonly used are:
• 5% CO2 in air
• 5% CO2, 5% O2 and 90% N2. This gas mixture allows for better embryo
development because it’s thought to be more similar to the condition found in
the oviduct and uterus
b. pH – the pH of the culture medium should be between 7.2 and 7.4. The pH can
be affected by temperature and gas phase if not properly buffered;
c. Osmolality – should be between 260 – 280 mOsm/kg;
d. Temperature – The culture temperature should be the same temperature found
by the embryo in the oviduct and uterus. In the cow, the normal temperature
ranges from 38.5 to 39oC and so this temperature should used during culture;
e. Water purity – resistivity of water must be less than 18 mOhm;
f. Sterility – procedures should be carried out taking care of keeping the culture
sterile, using sterile techniques and antibiotics if necessary;
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Bovine embryo culture procedure
1. Add 1000 μl HEPES-TALP in a microcentrifuge tube;
2. Place X-plate on the slide warmer and add ~5 ml of HEPES-TALP to each of the
wells;
3. After microscope and air have been warmed sufficiently, remove one 4-well plate
containing IVF drops from the incubator;
4. Remove oocyte-cumulus complexes (now called putative zygotes since many of
them have been fertilized; Figure 9) from each well of the 4-well plate and place
in the microcentrifuge tube. Up to 300 embryos can be loaded in one
microcentrifuge tube;
5. Repeat steps 3 and 4 until all plates have been processed;
6. Remove cumulus cells from putative zygotes by vortexing (Figure 10) the tube
containing the embryos/oocytes for 3-4 minutes;
Figure 9. Putative zygotes after 8-hour fertilization.
Note the cumulus cells detaching from the putative
zygote due to action of the sperm cells.
Figure 10. Vortexing COCs to remove cumulus
cells.
7. Two to three days after fertilization, embryos should be checked under a
microscope to determine the percentage that cleaved. This number is an
approximation of the percentage of oocytes that were fertilized. However it
should be noted that not only fertilized oocyte will cleave. Parthenotes are
unfertilized oocytes that due to external stimulus start to divide. The parthenotes
can reach blastocyst stage and when transferred to a cow, may survive for up to
30 days but will die due to failure in placentation and attachment to the uterus.
8. Seven or eight days after fertilization, embryos should be checked again and the
percentage of blastocysts formed recorded.
9. Grade 1 and grade 2 blastocysts are usually transferred to synchronized
recipients;
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Embryo culture in other species: further considerations
•
The growth rate of the embryo varies among species being very fast in the
murine and slower in the bovine.
.
Figure 11. Four-cell (top left) and 8-cell (top
right) bovine embryos (cell’s cytoplasms are
seen in red and nuclei in blue). Bovine
blastocyst on day 8 after fertilization (bottom).
Inner cell mass is seen as a compact group of
cells and the trophoblast as more disperse
cells
•
Embryonic growth requirement in vitro also varies greatly among species. For
instance, murine embryos can grow with minimum nutrients, if any at all, while
other species like bovine, porcine, equine and humans are much more selective
and sensitive to non-optimal conditions.
•
In most domestic species, embryos are transferred to the recipients at the morula
or blastocyst stage. In humans, however, is a common procedure to transfer the
embryos earlier, like at the 2-cell, 4-cell or 8-cell stages. This is to avoid the
negative effects that prolonged in vitro culture has on the embryo (growth arrest,
improper genome activation, large offspring syndrome). The drawback of this
procedure is the need to transfer multiple embryos to increase the chances of
establishing pregnancy which increases the chance of causing a multiple
pregnancy (twins, triplets, quadruplets or more).